The goal of this project is to understand the basic mechanisms by which transcription is regulated by sequence-specific transcription factors, in particular enhancer-binding proteins. Oncogenic transformation is usually the result of altered gene expression due to changes in transcriptional regulation. Indeed, many oncogenes encode transcriptional regulators that by being overexpressed or functionally altered in their DNA binding or activation properties become oncogenic. To study transcriptional regulation, we use as a model the SV40 early transcription unit, which depends on cellular activators to express the SV40 T and t tumor antigens. The SV40 early promoter contains a transcriptional enhancer composed of many redundant elements that respond to different cellular activators. One of these elements is the octamer motif ATGCAAAT, which is recognized by the two octamer motif- binding proteins Oct-1 and OCT-2. The octamer motif is a regulatory element found in both enhancers and proximal promoter regions, that displays both ubiquitous and cell-specific activities depending on the promoter context. These activities result in part from the ability of the ubiquitous Oct-1 and cell-specific Oct-2 proteins to differentially regulate transcription even though they possess the same DNA binding specificity. In this project, we will continue to use Oct-1 and Oct-2 as models to dissect the mechanisms of transcriptional regulation in human cells. We propose (i) to dissect how Oct-1 and Oct-2 stimulate transcription of small nuclear RNA genes that are transcribed by either RNA polymerase II (the U2 gene) or RNA polymerase III (the U6 gene); (ii) to investigate how Oct-1 and Oct-2 stimulate mRNA gene transcription; (iii) to ascertain the role of Oct-1 and Oct-2 phosphorylation in transcriptional regulation; and (iv) to elucidate the mechanisms of stimulation of transcriptional initiation by Oct-1 and Oct-2 using in vitro transcription assays. These studies will further our understanding of the mechanisms of transcriptional regulation and will form a basis to understand how modification of these regulatory mechanisms can lead to oncogenesis.